Birth and Death of Proteins

protein turnover purpose

• to rapidly degrade misfolded, damaged, or oxidized proteins before they assemble into toxic aggregates

• Maintain cellular health, regulate proteins levels, and allow the body to adapt to changing needs 

• Regulates the timing of cell cycle transitions and signaling cascades by removing transient regulatory proteins (cyclins)

Role of chaperones

• Bind to exposed hydrophobic regions to incomplete folded proteins and help them bind. Not spontaneous process.

• Assist, Rescue, Protect

• Often require many cycles of ATP hydrolysis

Hsp 60 and Hsp 70

• heat shock proteins

• produced in response to heat, toxins, infection or other cellular damage

• play a critical role in maintaining homeostasis by assisting in the correct folding of proteins, preventing misfolding, and facilitating the degradation of damaged proteins

TEST QUESTION: Hypothesis of infectious nature of proteins

infected prions activate as an enzyme that coverts normal prions into infected prion

Converts the alpha helices in the normal protein to beta sheets, that when stacked, can form amyloid fibrils

Examples of Protein Degradative Signals

• PEST – rich sequences – high percentage of proteins that have very short half-lives in cells exhibit short peptides sequence enriched in Pro-Glu-Ser-Thr 

• N-terminal residues that are very basic or hydrophobic, called the N-end rule

• Hydrophobic patch on protein’s surface 

Ubiquitin

• Small, highly conserved protein in all eukaryotic cells that can covalently attach to lysines of other proteins. Attachment of a short chain of ubiquitins to such a lysine can tag a protein for intracellular proteolytic destruction by proteasome.

• Marks proteins for degradation or alter trafficking

Ubiquitin-Proteasome System (UPS)

• E1 (Ubiquitin Activating Enzyme): Uses Atp hydrolysis (energy) to transfer ubiquitin to the active site of E1 (ubiquitin activates the enzyme )

• E2 (Ubiquitin-Conjugating Enzyme): Ubiquitin is transferred to the active site of E2

• E3 (Ubiquitin Ligase): Interacts with both E2 and the target protein. Ubiquitin is transferred from E2 to lysine residues of the target protein

(target protein binds E3 and ubiquitin chain is added) 

****this is the only enzyme that recognizes the signal of ubiquitin. The other two set it up

The tagged protein is recognized and degraded by the proteasome

E1 (Ubiquitin Activating Enzyme)

Uses Atp hydrolysis (energy) to transfer ubiquitin to the active site of E1 (ubiquitin activates the enzyme)

E2 (Ubiquitin-Conjugating Enzyme)

Ubiquitin is transferred to the active site of E2

E3 (Ubiquitin Ligase)

• Interacts with both E2 and the target protein. Ubiquitin is transferred from E2 to lysine residues of the target protein

• (target protein binds E3 and ubiquitin chain is added) 

****this is the only enzyme that recognizes the signal of ubiquitin. The other two set it up

Final step of the UPS pathway

The tagged protein is recognized and degraded by the proteasome

What does the Proteasome do?

breaks down and recycles proteins that are damaged, misfolded, or no longer needed.

How does the proteasome digest ubiquitin tagged proteins

1. Recognition: A protein is tagged with a chain of ubiquitin molecules, which serves as a signal for degradation. The proteasome recognizes the ubiquitin tag.

2. Unfolding: The target protein is removed from ubiquitin and unfolded before being fed into the proteasome.

3. Entry into proteasome: The unfolded protein is threaded into the central cylinder chamber of the proteasome.

4. Digestion: Proteases located inside the central chamber cleave the protein into small peptide fragments.

5. Ubiquitin is recycled: The ubiquitin molecules are released and can be reused to tag other proteins.

How is protein degradation regulated

• by controlling which proteins receive ubiquitin tags.

• In response to intracellular or environmental signals, a degradation signal can be created on a target protein.

• Ubiquitin ligases, particularly E3 ligases, recognize the target and facilitate its ubiquitination, marking it for degradation by the proteasome.

• E3 ligases can themselves be activated by phosphorylation or allosteric changes.

Prions

misfolded, infectious proteins that cause fatal neurodegenerative diseases

** they exist naturally

Prion Protein (PrP^c)

• glycoproteins found mainly on the surface of neurons

• Exact function is not known, but helps with myelin maintenance, cell signaling, neuroprotection, and synaptic function

Prp*/ PrP^Sc

abnormal, misfolded form of prion protein

Scrapie Agent

• Prion disease in sheep that can be passed from mother to lamb

• Initially assumed to be a virus, but none was found. Agent was also resistant to nucleases

• Agent was later identified as a prison

amyloid fibril

• Formed by already abnormal prions

• Convert normally folded molecules to more pathological prions

• *** positive feedback loop

• Stable structures

• Most resistant to chemical agents, radiation, and high temperaures

• Why is the brain vulnerable to protein aggregates

Why is the brain vulnerable to protein aggregates

• Quality control mechanism governing proteins gradually declines with age, which may cause protein aggregates

• There is evidence of infections: prions preventing UPS degradation by interactions w proteasome cap

• The brain also has highly organized nerve cells that can’t regenerate, and is therefore vulnerable to damage

How does an infectious prion spread in the body?  

1. Inherited: Mutation in prion gene (FFI)

2. Sporadic: Normal prion misfolds to abnormal prion

3. Acquired: results from exposure ot PrPSc from an outside source

Mad Cow disease

• Gov name is BSE (bovine spongiform encephalopathy) - progressive neurological disorder of cattle caused by prions 

• Speech, coordination, and behavior decline

• Passed between cows through recycling animal carcasses for meat and bone meal protein, which was fed back to the cattle

Prevention for Mad Cow Disease

• FDA banned the use of mammalian protein in animal feed

• Brain and spinal cord tissues couldn’t be processed for food

thalamus

FFI (Fatal Familial Insomnia, destroys the ability to sleep affects what party of the brain

cerebral cortex

CJD (Creutzfeldt-Jakob disease) affects what party of the brain

Brain stem

Mad Cow/ BSE (bovine spongiform encephalopathy) affect what part of the brain

Kuru

Caused by consuming prion‑infected human brain tissue during funerary cannibalism, with symptoms such as tremors, ataxia, and progressive neurodegeneration, and no available cure.

cerebellum

Kuru affects what part of the brain

Variant Creutzfeldt Jakob disease

• Neurodegenerative brain disorder

• Evidence that CJD and BSE (mad cow disease) are cause by the same prion

Symptoms of Creutzfeldt Jakob disease

• Psychiatric behavioral symptoms

• Impaired speech, vision, movement, memory, coordination, etc.

• Rapid deterioration (months)

Diagnosis of Creutzfeldt Jakob disease

• Happens the fastest; they die within the year

• Detectable 14-3-3 protein in the cerebrospinal fluid 

• Can only be confirmed post-mortem by the presence of aggregates in brain tissue

Treatment of Creutzfeldt Jakob disease

• No real  cure available

• Some antibodies can be used against prions

• Molecular tools that are delivered to the brain and add a chemical tag to the gene for the prion protein to stop the protein from being produced

TEST QUESTION: Hsp 60 and GroES caps

Essentially, this protein forms an isolated, hydrophilic chamber for the protein. The protein enters the hydrophilic interior and then ATP is utilized to add a GroES cap. This cap prevents the protein from leaving; multiple cycles of ATP open and close the cap until the protein is folded properly and is able to leave.

TEST QUESTION: Destruction via the Ubiquitin Proteasome System.

Ubiquitin-activating enzyme (E1) utilizes ATP to add ubiquitin to its active site which it then transfers to ubiquitin-conjugating enzyme (E2). Then, ubiquitin ligase pulls together E2 and the target protein by recognizing PEST signals to attach the ubiquitin to the lysine residue. Once 4 ubiquitin are added to the target protein, it is recognized by a proteasome complex which is lined with proteases that break the polypeptide into amino acids.